Fictive Gill and Lung Ventilation in the Pre- and Postmetamorphic
Tadpole Brain Stem
C. S. TORGERSON, M. J. GDOVIN, AND J. E. REMMERS
1
Respiratory Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada; and
2
Division of Life Sciences, University of Texas at San Antonio, San Antonio, Texas 78249
INTRODUCTION
The assumption of terrestriality by vertebrates required
profound changes in the form and function of the respiratory
system associated with the transition from water to air (see
reviews by Dejours 1988; Gans 1970; Little 1983; Randall
et al. 1981; Shelton et al. 1986). Amphibians, modern descendents of the early tetrapods that migrated from water to
land, developed complex respiratory processes and structures
in response to differences in the physical properties of air
and water. This is particularly apparent in larval forms where
gas exchange occurs at multiple sites (skin, gills, and lungs),
allowing simultaneous exploitation of both respiratory media. Investigation of the ontogeny of respiratory rhythm gen-
eration in the tadpole provides a unique opportunity to examine the development of basic neuronal processes that generate and regulate respiratory motor output.
The gas exchange needs of very immature bullfrog larvae
are met by the passive diffusion of gases across the body
wall (Burggren 1984). As body mass and metabolic demand
increase, tadpoles require progressively more efficient gas
exchange organs (gills and lungs). The gills are ventilated
by a constant, unidirectional stream of water drawn into the
mouth, through the oropharyngeal cavity, and out of the
opercular spout by rhythmic dorsoventral movement of the
buccal floor (DeJongh 1968). By contrast, tidal flow of air
into and out of the paired, saclike lungs is accomplished by
the same buccal musculature, drawing air in the oropharynx
and forcing it into the lungs (Burggren 1984). Lung inflation
occurs during the brief opening of the glottis and remains
full until the beginning of the next cycle when exhalation,
powered by elastic recoil of the respiratory system, drives
the air out of the lungs. Before onset of metamorphosis
(Taylor-Köllros stage 16), ventilatory patterns appear homologous to those described for lungfish, regular branchial
movements pumping water over the gills and interspersed
with irregular lung breaths (McMahon 1969; Smatresk
1990). During metamorphic climax (stage 18–19), the gills
and tail degenerate and the lungs become the dominant site
for O2 exchange, reaching full maturity in stages 20–25
(Burggren and West 1982). On completion of metamorphosis, the forelimbs develop, the gills and tail are completely
reabsorbed, and the branchial cavity is filled with air between
breaths (Smatresk 1990).
Investigation of the neural mechanisms responsible for respiratory neuromuscular activity in amphibians was facilitated
by the development of superfused in vitro brain stem–spinal
cord preparations. The functional significance of the patterns
of spontaneous bursting activity observed in cranial nerve
(CN) roots of the completely isolated adult frog brain stem
preparations (McLean et al. 1995a,b) was elucidated by correlating CN root activity with activity of nerves to respiratory
muscles in an in situ preparation (Kimura et al. 1997) and
with neurograms, electromyograms, and mechanical events
related to oropharyngeal and pulmonary ventilatory cycles
from in vivo preparations (DeJongh and Gans 1969; Ito and
Watanabe 1962; Kogo et al. 1994a; Kogo and Remmers
1994b; Sakakibara 1984a,b; West and Jones 1975). Similarly,
recent studies of in vitro tadpole brain stem preparations began
to characterize the patterns of respiratory activity related to
gill and lung ventilation (Galante et al. 1996; Liao et al.
0022-3077/98 $5.00 Copyright q 1998 The American Physiological Society
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Torgerson, C. S., M. J. Gdovin, and J. E. Remmers. Fictive gill
and lung ventilation in the pre- and postmetamorphic tadpole brain
stem. J. Neurophysiol. 80: 2015–2022, 1998. The pattern of efferent neural activity recorded from the isolated brain stem preparation of the tadpole Rana catesbeiana was examined to characterize
fictive gill and lung ventilations during ontogeny. In vitro recordings from cranial nerve (CN) roots V, VII, and X and spinal
nerve (SN) root II of premetamorphic tadpoles showed a coordinated sequence of rhythmic bursts occurring in one of two patterns,
pattern1, high-frequency, low-amplitude bursts lacking corresponding activity in SN II and pattern 2, low-frequency, highamplitude bursts with coincident bursts in SN II. These two patterns
corresponded to gill and lung ventilatory burst patterns, respectively, recorded from nerve roots of decerebrate, spontaneously
breathing tadpoles. Similar patterns were observed in brain stem
preparations from postmetamorphic tadpoles except that they
showed a greater frequency of lung bursts and they expressed
fictive gill ventilation in SN II. The laryngeal branch of the vagus
(Xl) displayed efferent bursts in phase with gill and lung activity,
suggesting fictive glottal constriction during gill ventilation and
glottal dilation during lung ventilation. The fictive gill ventilatory
cycle of pre- and postmetamorphic tadpoles was characterized by a
rostral to caudal sequence of CN bursts. The fictive lung ventilatory
pattern in the premetamorphic animal was initiated by augmenting
CN VII discharge followed by synchronous bursts in CN V, X,
SN II, and Xl. By contrast, postmetamorphic patterns of fictive
lung ventilation were characterized by lung burst activity in SN II
that preceded burst onset in CN V and followed the lead burst in
CN VII. We conclude that recruitment and timing of pattern 1 and
pattern 2 rhythmic bursts recorded in vitro closely resemble that
recorded during spontaneous respiratory behavior, indicating that
the two patterns are the neural equivalent of gill and lung ventilation, respectively. Further, fictive gill and lung ventilatory patterns
in postmetamorphic tadpoles differ in burst onset latency from
premetamorphic tadpole patterns and resemble fictive oropharyngeal and pulmonary burst cycles in adult frogs.
2016
TABLE
C. S. TORGERSON, M. J. GDOVIN, AND J. E. REMMERS
1.
Mean latency and time to peak of pattern 1 and 2 bursts from CN V, VII, X, SN II, and X
Premetamorphic Stages
Postmetamorphic Stages
Pattern 1
Pattern 2
Nerve
n
Latency
Time-to-peak
Latency
CN V
CN VII
CN X
SN II
CN Xl
5
10
6
7
3
0481.4 { 73.7*
0.0†
454.3 { 53.8*†
NA
090.4 { 42.5
367.7 { 39.9
473.1 { 45.5
472.5 { 91.6
NA
496.9 { 142.0
232.3 { 79.5*
0.0
286.5 { 16.1*
324.3 { 59.5*
231.8 { 124.7*
Pattern 1
Time-to-peak
n
Latency
{
{
{
{
{
5
6
5
5
0416.1 { 81.3*
0†
388.6 { 19.8*†
541.9 { 35.4*†
547.0
603.9
503.1
378.4
209.1
127.3
98.1
91.5
88.8
58.4
Pattern 2
Time-to-peak
Latency
{
{
{
{
394.2 { 50.6*
0.0
320.7 { 38.7*
246.1 { 34.1*†
324.2
628.0
360.4
471.5
24.3*
41.8
31.6*
28.8*
Time-to-peak
444.5
755.8
404.1
549.5
{
{
{
{
61.3*
36.2
39.1*
30.3*
Data shown as means { SE in ms. Onset latency of pattern 1 and 2 bursts calculated with respect to CN VII. SN II displayed no activity (NA) during fictive gill ventilation in premetamorphic tadpoles.
Bursting activity of Xl was measured in premetamorphic tadpoles only. * Significantly different from CN VII mean values within both developmental group and type of ventilation (P õ 0.05). † Significantly
different from CN V mean values within both developmental group and type of ventilation (P õ 0.05).
ventilatory motor output. To achieve these two goals, we
recorded efferent activity from CN roots, the second spinal
nerve (SN) root and the laryngeal branch of the vagus (Xl)
in pre- and postmetamorphic tadpoles. SN II innervates the
hypoglossal muscles and was shown to be a marker of lung
breaths in the metamorphic tadpole in vivo (Gdovin et al.
1998). Xl can be considered a respiratory nerve because its
efferent activity controls the precise opening and closing of
the glottal valve during lung ventilation.
METHODS
General
Experiments were performed on 17 larval bullfrog tadpoles (R.
catesbeiana) of either sex, obtained from a commercial supplier
(Charles D. Sullivan, Nashville, TN). Specimens were assigned
to one of two groups based on the criteria of Taylor and Köllros
(1946), premetamorphic (stages 4–14, n Å 10,) and postmetamorphic (stages 20–22, n Å 7). During the 5 days preceding experimentation, all tadpoles were housed in aerated, filtered aquariums
FIG . 1. Moving time average of cranial
nerve (CN) V, VII, and X showing patterns
1 and 2 from the isolated brainstem preparation of a stage 11 Rana catesbeiana tadpole superfusate with artificial cerebrospinal fluid (CSF) having a PCO2 of 45 Torr
and pH of 7.4. The height of the respiratory
bursts was measured by using arbitrary
units.
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1996; Pack et al. 1993; Torgerson et al. 1997a). However,
the separate identification of lung and gill ventilatory activities
in these studies must be considered tentative, as an arbitrary
CN amplitude criterion alone was used to identify putative
gill and lung bursts in CN roots, i.e., large amplitude bursts
were assumed to be fictive lung breaths, and small amplitude
bursts were assumed to be fictive gill bursts. If the isolated
tadpole brain stem preparation is to be utilized as a tool to
investigate fundamental questions of rhythmogenesis and central chemoreception, fictive gill and lung ventilatory activity
must be separately identified.
The primary purpose of this study is to describe the spatial
and temporal patterns of spontaneous bursting activity characteristic of fictive gill and lung ventilation in the in vitro
tadpole brain stem. In a separate article (Gdovin et al. 1998),
we describe the motor output of the spontaneously breathing
larval Rana catesbeiana. In this study, we provide data from
the isolated tadpole brain stem preparation that can be correlated with observations from the in vivo study. A second
purpose is to describe ontogenetic changes in gill and lung
ONTOGENY OF FICTIVE BREATHING IN THE TADPOLE BRAIN STEM
(19–217C) and fed TetraFin Staple Food (TetraWerke, Germany).
The Animal Care Committee of the University of Calgary, Canada,
approved the experimental protocol used in these studies.
Surgical preparation
cordings described by Gdovin et al. (1998). We define the burst
frequency to be the number of bursts per unit time. To evaluate
developmental differences in respiratory bursting patterns from CN
V, VII, X, SN II, and Xl, we measured the time to peak and relative
time of onset of fictive gill and lung ventilatory bursts for each
10-min recording at pH 7.4. Mean time to peak was calculated
from the onset of the burst to the time of peak amplitude, and
mean latency of fictive gill and lung ventilation was determined
for each nerve with respect to the onset of the CN VII burst. Mean
values of gill and lung motor output for each animal were then
used to calculate group means { SE. Significant differences in the
latency and time to peak of fictive gill and lung ventilation within
and between each developmental group were examined with a oneway analysis of variance (ANOVA) for repeated measures, with
the criterion of statistical significance at P õ 0.05. A Student–
Newman–Keuls test of pairwise multiple comparisons was used
to test significant differences among nerves within treatment groups
when ANOVA revealed significant treatment effects.
RESULTS
Rhythmic, coordinated bursting patterns were recorded
from CN V, VII, X, SN II, and Xl in premetamorphic (n Å
10) and postmetamorphic (n Å 6) larvae when superfused
Recording chamber
The brain stem was transferred to a superfusion recording chamber that was described previously (Torgerson et al. 1997b). Briefly,
two superimposed disks (2.5-cm OD, 1-mm thickness) with central
elliptical holes (2.0 1 0.5 cm) partitioned the chamber into upper
and lower compartments. Fine netting (1.0-mm2 mesh) spanning
the holes was attached to the upper surface of the upper and lower
disks, thereby creating a space between the netting for the brain
stem. The brain stem was positioned between the two nets, ventral
side up, and superfused with artificial CSF (22–247C) equilibrated
with a mixture of CO2-O2 . The superfusate was conducted from
the opposite end of the upper compartment via a paper wick.
Recording
Efferent recordings were obtained from the roots of CN V, VII
and X, SN II (hypoglossal in the adult), and Xl by using suction
electrodes. The pipettes were made from 1-mm OD, thin-walled
borosilicate glass and then pulled to a fine tip with a horizontal
micropipette puller (Brown-Flaming, model P80). The tip was
broken and beveled (Shöhli, Lapp-Technik) to achieve various
inner tip diameters ranging from 90 to 350 mm. Action potentials
were amplified (AM Systems no.1700, Tektronix AM 502), filtered (100 Hz to 1 kHz), and recorded on videotape with a pulse
code modulator (Neurodata DR-890). The signals were simultaneously averaged with a Paynter time averager, displayed on a polygraph (Gould), and digitized and analyzed on a Pentium PC (Datapac II software).
Equilibration of the superfusate with gas having a PCO2 of 45
and 17 Torr (balance O2 ) produced pH values of 7.4 and 7.8. After
the brain stem was superfused in the recording chamber for ¢60
min (pH 7.8), stable nerve activities were recorded for 10 min at
pH 7.8 and 7.4. Between each 10-min recording, a 5-min equilibration period ensued to allow equalization of tonometer and recording
chamber pH and stabilization of the brain stem response to the
new superfusate pH.
Analysis
As described in RESULTS, gill and lung bursts were identified by
SN II amplitude criteria and by correlations with the SN II re-
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FIG . 2. Unprocessed and integrated neurograms of CN V, VII, and X
recorded from a premetamorphic (stage 4) tadpole brain stem preparation
superfused with artificial CSF having a PCO2 of 45 Torr and pH of 7.4. In
raw neurograms, the recruitment of new motor units during pattern 2 is
clearly distinguishable. The height of the respiratory bursts was measured
by using arbitrary units.
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Tadpoles were anesthetized in tricaine methane sulfonate
(1:10,000) and then weighed. Once unresponsive, the dorsal cranium was removed, and with the aid of a dissecting microscope
the cranial and SNs were severed at their respective ostia. After
removing the dura and arachnoid dorsally and ventrally, the brain
stem was transected just caudal to the level of the fourth SN and
just rostral to CN V. In a subset of premetamorphic tadpoles
(n Å 3), Xl, which innervates glottal constrictors and dilators, was
dissected and left attached to the brain stem. In these animals, the
main trunk of the vagus was freed dorsally by resection of the
cartilage surrounding the postotic foramen and by careful excision
of the dorsolateral margin of the semicircular canal. Using a ventral
approach, Xl was exposed by excising the overlying tissue and
isolating the entire nerve. Throughout the dissection, the brain stem
was superfused with a bicarbonate containing artificial cerebrospinal fluid (CSF) of the following composition (in mM): 104 NaCl,
4 KCl, 1.4 MgCl2 , 10 D-glucose, 25 NaHCO3 , and 24 CaCl2 ,
bubbled with 98% O2-2% CO2 , pH 7.8, corresponding to the
plasma pH of anuran amphibians (West et al. 1987).
2017
2018
C. S. TORGERSON, M. J. GDOVIN, AND J. E. REMMERS
FIG . 3. Integrated neurograms of CN VII and
spinal nerve (SN) II showing patterns 1 and 2
recorded from a postmetamorphic (stage 21) tadpole brain stem preparation superfused with artificial CSF having a PCO2 of 45 Torr and pH of 7.4.
The height of the respiratory bursts was measured
by using arbitrary units.
tadpole larvae. Premetamorphic fictive ventilation was characterized by pattern 1 bursts observed in CN VII and X,
punctuated by sporadic, usually isolated, pattern 2 bursts
appearing in all three nerve recordings. Postmetamorphic
fictive ventilation, by contrast, was distinguished by an increase in the frequency of pattern 2 bursts in CN VII, X,
Bursting patterns
As illustrated in Fig. 1, integrated neurograms of CN V,
VII, and X from a premetamorphic tadpole revealed two
different patterns of rhythmic activity, high burst frequency,
low-amplitude bursts (pattern 1) and low burst frequency,
high-amplitude bursts (pattern 2). Figure 2 shows both unprocessed and integrated neurograms of CN V, VII, and X
in a premetamorphic (stage 4) tadpole, demonstrating patterns 1 and 2. Examination of action potentials in all three
nerve records indicates the recruitment of new motor units
during pattern 2, as described by Gdovin et al. (1998), further substantiating the distinction between the two motor
output patterns.
Commonly, patterns 1 and 2 were not clearly distinguishable from the amplitude of bursts of CN roots. An example
of such is illustrated in Fig. 3, where differences between
the peak amplitude of patterns 1 and 2 in CN VII were
minimal. However, the large amplitude SN II burst during
pattern 2 provided an unequivocal marker for this pattern and
allowed ready distinction between the two fictive ventilatory
patterns. Because of the unequivocal nature of SN II bursts
and their direct correlation to lung ventilation, as reported
by Gdovin et al. (1998), we used SN II as the marker of
pattern 2, whenever possible, in our analysis. Similarly, the
Xl neurogram demonstrated pronounced bursting during pattern 2 but only modest activity during pattern 1, as shown in
Fig. 4. This distinctive behavior of SN II and Xl neurograms
during pattern 2 was consistently observed in all recordings.
In 3 of 10 premetamorphic animals and 1 of 6 postmetamorphic larvae, adequate recordings were not available from SN
II. Accordingly, an amplitude criterion was used to identify
pattern 2 bursts in the analysis shown in Table 1.
The pattern of respiratory motor output was dependent on
developmental stage. Figure 5 shows typical simultaneous
integrated neurograms of CN VII, X, and SN II from premetamorphic (stage 12) and postmetamorphic (stage 22)
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FIG . 4. Integrated neurograms of CN VII, Xl, and SN II showing patterns 1 and 2 recorded from a premetamorphic (stage 13) tadpole brain
stem preparation superfused with artificial CSF having a PCO2 of 45 Torr
and pH of 7.4. The height of the respiratory bursts was measured by using
arbitrary units.
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with artificial CSF of pH 7.8 and 7.4. Although simultaneous
recordings of all nerves were not successful in all animals,
rhythmic bursts were recorded in two or more of the nerves
in each experiment. Table 1 provides the number of animals
in which each nerve as successfully recorded.
ONTOGENY OF FICTIVE BREATHING IN THE TADPOLE BRAIN STEM
2019
and SN II, occurring predominantly in clusters, and by the
emergence of a pattern 1 in SN II neurograms.
Latency and time-to-peak of bursts
FIG . 6. Onset latency and time to peak profiles of pattern 1 and 2 bursts
in pre- (A) and postmetamorphic (B) tadpole larvae. Mean time to peak
represented by the relative length of the bars with SEs plotted on the right.
Mean onset burst latency determined with respect to CN VII (outlined by
gray) and indicated by the relative horizontal position of bars with standard
errors shown on the left. * Significantly different from CN VII mean values
within both developmental group and type of ventilation (P õ 0.05);
† significantly different from CN V mean values within both developmental
group and type of ventilation (P õ 0.05).
FIG . 5. Integrated neurograms of CN VII, X, and SN II recorded from
premetamorphic (stage 12, A) and postmetamorphic (stage 22, B) tadpole
brain stem preparations superfused with artificial CSF having a PCO2 of 45
Torr and pH of 7.4, showing transition in the pattern of fictive ventilation
with development. The height of the respiratory bursts was measured by
using arbitrary units.
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V, VII, X, and SN II bursts all differed significantly (P õ
0.05) with respect to each other. The time to peak of CN VII
bursts was significantly greater (P õ 0.05) than the time to
peak in CN V, X, and SN II bursts, and the time to peak of
SN II burst activity was significantly greater (P õ 0.05) than
that of CN V and X. Further, the time to peak of CN VII
pattern 1 bursts of postmetamorphic larvae was significantly
greater (P õ 0.05) than that of premetamorphic tadpoles.
Like premetamorphic patterns, pattern 2 in postmetamorphic
tadpoles was initiated by augmenting activity in CN VII. However, after preliminary CN VII onset, postmetamorphic pattern
2 cycles were characterized by sequential bursting of SN II,
followed by simultaneous onset of CN V and X activities (Fig.
6B, open bars). With respect to the mean latency of burst
onset, CN VII led CN V, X, and SN II by a significant interval
(P õ 0.05), and SN II significantly preceded (P õ 0.05) CN
V. The time to peak of pattern 2 in CN VII was significantly
longer (P õ 0.05) than that observed in CN V, CN X, and
SN II of postmetamorphic larvae.
DISCUSSION
Recordings of efferent activity from the roots of CN V,
VII, X, SN II, and from the respiratory nerve Xl in the
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PREMETAMORPHIC ANIMALS. Mean time to peak and latency
values of patterns 1 and 2 are displayed in Fig. 6 and listed in
Table 1. The pattern 1 ventilatory cycle of premetamorphic
tadpoles was characterized by sequential bursts in CN V,
VII, and X roots, with activity in Xl occurring in phase with
CN VII (Fig. 6A, solid bars). Onset latency of CN V, VII,
and X differed significantly (P õ 0.05) from each other as
well as the onset of Xl in comparison with CN V and CN
X. By contrast, pattern 2 cycles in premetamorphic tadpoles
began with slowly augmenting CN VII discharge that burst
synchronously with CN V, X, SN II, and Xl (Fig. 6A, open
bars). The onset of the pattern 2 bursts in CN V, X, Xl, and
SN II significantly lagged behind (P õ 0.05) the onset of
lung activity in CN VII. Premetamorphic tadpoles showed
no significant difference (P ú 0.05) in the time to peak of
cranial and SN bursts when comparisons were made within
pattern 1 and 2 ventilatory cycles.
POSTMETAMORPHIC ANIMALS. Pattern 1 in postmetamorphic
larvae was distinguished by the emergence of SN II discharge
that burst after sequential activation of CN V, VII, and X
(Fig. 6B, filled bars). The relative mean onset latency of CN
2020
C. S. TORGERSON, M. J. GDOVIN, AND J. E. REMMERS
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lung bursts recorded in vitro ranged between 10 and 100%
of in vivo burst time to peak measurements, correlation between both preparations was close, in view of in vitro peripheral deafferentation and gas/pH tissue status (Torgerson et
al. 1997b).
Although the spatiotemporal characteristics of pattern 1
and 2 bursts appear similar in pre- and postmetamorphic
larvae, several important distinctions can be made. In premetamorphic larvae ( õstage 16), respiratory neural output
consisted predominantly of gill activity interrupted occasionally by isolated lung bursts. This finding corresponds with
respiratory patterns described in intact premetamorphic tadpoles (Burggren and Doyle 1986; Infantino 1992) and confirms previous results from isolated premetamorphic tadpole
brain stem preparations (Torgerson et al. 1997a).
Premetamorphic gill motor output cycles in vitro were
initiated by CN V activity and followed by sequential bursts
of equal time to peak in CN VII and X. This finding agrees
with Gradwell’s (1972a,b) description of gill irrigation mechanics in premetamorphic tadpoles, showing coordination
of mouth opener and closer muscles (innervated by CN V),
buccal floor elevators and constrictors (innervated by CN
VII), and pharyngeal cavity constrictors and dilators (innervated by CN X). In the isolated premetamorphic tadpole
brain stem preparation, we detected no bursting activity in
SN II during fictive gill ventilation. Although Gradwell
(1972a,b) attributed the activation of mouth opener and pharyngeal constrictor muscles to SN motor output, the contribution of these muscles to gill ventilation was minimal based
on electromyographic and pressure recordings. Further, our
results agree with Taylor (1985), who reported a similar
rostral-to-caudal bursting sequence in CN output during gill
ventilation in studies of dogfish. Finally, we observed rhythmic activity in Xl during fictive gill ventilation that burst in
phase with CN VII. Xl innervates dilator and constrictor
muscles of the glottis (Sakakibara 1984a) and was previously shown to display bursting activity during fictive oropharyngeal ventilation in in vivo (Kogo et al. 1994a,b) and
in situ (Kimura et al. 1997) adult bullfrog preparations. Xl
activity in premetamorphic larvae during fictive gill ventilation may represent fictive glottal closure, ensuring that no
water enters the lungs before being forced over the gills.
Fictive lung ventilatory cycles in premetamorphic tadpoles were led by CN VII, demonstrating biphasic activity,
with a preliminary slowly augmenting discharge followed
by an abrupt increase in activity synchronous with CN V,
X, Xl, and SN II. Our recordings of Xl activity, showing a
burst of activity coincident with synchronous lung bursts in
CN V, X, and SN II, suggest that laryngeal muscle activity
is modulated during gill and lung bursts. This agrees with
results reported by Kogo et al. (1994a,b) and Kimura et al.
(1997) in the adult frog. Further, exclusive lung burst activity in SN II provides a specific and sensitive marker of fictive
lung activity in premetamorphic tadpole, allowing clear differentiation from gill motor output.
Although no study has systematically described the mechanics of lung ventilation in larval amphibians, the basic
mechanism, coordinated by buccal and pharyngeal musculature normally used to propel water through the branchial
chambers, is thought to resemble lung ventilation described
in lung fish (McMahon 1969) and adult frogs (DeJongh and
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isolated brain stem preparation of larval R. catesbeiana displayed two types of rhythmic bursting: pattern 1, a highfrequency, low-amplitude oscillation in a rostral-to-caudal
sequence, and pattern 2, a low-frequency, high-amplitude
rhythm initiated by CN VII and lacking the rostral-to-caudal
progression (Figs. 1–5). Pattern 1 was virtually identical in
pre- and postmetamorphic animals with the exception that
the latter included an oscillation in SN II. Pattern 2 was
similar in pre- and postmetamorphic larvae but exhibited
distinct differences in the burst latency between nerves.
These patterns bear a striking resemblance to those recorded
by Gdovin et al. (1998) from CN V, VII, and SN II in the
spontaneously breathing decerebrate tadpole, where gill and
lung ventilation were mechanically defined. Because of their
close correspondence to the in vivo patterns, we classify
pattern 1 as fictive gill ventilation and pattern 2 as fictive
lung ventilation.
Previous studies used an arbitrary CN amplitude criterion
for identifying gill and lung bursts (Galante et al. 1996; Liao
et al. 1996; Pack et al. 1993; Torgerson et al. 1997a). The
use of such a criterion was not validated, and its sensitivity
and specificity are suspect. This study elaborates on the work
of Pack et al. (1993) by describing the use of high-amplitude
SN II bursts as a sensitive marker of fictive lung ventilation.
This study also describes correlates of fictive gill and lung
ventilation such as the spatiotemporal characteristics as well
as recruitment of Xl bursts during fictive lung ventilation.
In our experience, CN burst amplitude is not adequate to
identify fictive gill and lung bursts in many cases, but, when
used in combination with SN II, any particular burst can be
unequivocally identified as pattern 1 or pattern 2.
The overall burst frequencies of pattern 1 and pattern 2
closely resemble those previously reported by Torgerson et
al. (1997b) in pre- and postmetamorphic tadpoles. Similarly,
the frequency and rostral-to-caudal sequence of pattern 1
observed here closely resembles those reported by Gdovin
et al. (1998) for gill ventilation. Comparison of Fig. 6 from
Gdovin et al. (1998) and Fig. 6 from this paper show both
fictive gill cycles initiated by bursts in CN V followed by
activity in CN VII. Further, the frequency of gill bursts in
decerebrate metamorphic tadpoles during normoxia (49.3 {
6.1 min 01 ) (Gdovin et al. 1998) correlates with gill burst
frequency (43.0 { 2.7 min 01 ) previously measured in the
isolated metamorphic tadpole brain stem preparation when
superfused with artificial CSF of pH 7.8 (Torgerson et al.
1997a). This evidence supplements the recruitment and amplitude data discussed above and leads to the conclusion
that low-amplitude, high-frequency bursts in vitro represent
fictive gill ventilation.
Gdovin et al. (1998) characterized fictive lung ventilation
by synchronous burst activity in CN VII and V and by the
recruitment of SN II. Similarly, we observed simultaneous
bursting in CN V and VII and the appearance of SN II bursts
during pattern 2 in vitro, suggesting that high-amplitude,
low-frequency bursts represent fictive lung activity. Fictive
lung ventilation frequency, previously described in the isolated metamorphic brain stem preparation (0.2 { 0.1 min 01 ,
artificial CSF, pH 7.8) (Torgerson et al. 1997a), matched
measurements reported by Gdovin et al. (1998) in the decerebrate metamorphic tadpole under normoxic conditions
(0.3 { 0.1 min 01 ). Although the time to peak of gill and
ONTOGENY OF FICTIVE BREATHING IN THE TADPOLE BRAIN STEM
/ 9k2d$$oc11 J819-7
cles in postmetamorphic tadpoles were initiated by augmenting CN VII activity that burst simultaneously with CN
V and X. By contrast, the onset of postmetamorphic lung
burst activity in SN II preceded burst onset in CN V and X,
shifting toward CN VII. This pattern resembles the phase
relations of lung burst activity reported by Kogo et al.
(1994a,b), McLean et al. (1995a,b), and Kimura et al.
(1997) in the adult frog, demonstrating similarity between
postmetamorphic and adult lung ventilatory cycles.
Overall, cranial and SN activity from the isolated brain
stem of larval R. catesbeiana closely resembles the pattern
of neural activity during gill and lung ventilation in the
spontaneously breathing decerebrate tadpole. Recordings of
SN II bursts improve the accuracy of distinguishing fictive
lung from gill ventilation and in many cases are essential
for separating these two types of ventilatory motor outputs.
Fictive gill and lung ventilatory patterns in postmetamorphic
tadpoles differ in burst onset latency from premetamorphic
tadpole patterns and resemble fictive oropharyngeal and pulmonary burst cycles in adult frogs. Thus, in addition to establishing a descriptive framework for investigating the ontogeny of neural-respiratory control in vitro, we demonstrate a
shift in the pattern of gill and lung motor output that accompanies the transition from water to air breathing and provides
new insight into the origin and development of the amphibian respiratory CPG.
This work was supported by the Alberta Heritage Foundation for Medical
Research and the Medical Research Council of Canada.
Address for reprint requests: J. E. Remmers, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1,
Canada.
Received 7 October 1997; accepted in final form 8 June 1998.
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